The effect of a single dose of cyclophosphamide (CY) on delayed type hypersensitivity (DH) and acquired cellular resistance (ACR) to Listeria monocytogenes infection in mice was studied. Intraperitoneal or intracutaneous immunization with L forms of L. monocytogenes did not result in protection against lethal challenge. A positive DH could be observed when CY-treated mice were intracutaneously immunized with 10(8) or more L forms. Intraperitoneal injection of viable L. monocytogenes resulted only in a narrow dose range in survival on immunization and partial protection on challenge. Protection was accompanied by DH. Intracutaneous injection of Listeria in Freund's complete adjuvant permitted the use of even 10(9) viable bacteria for immunization. This figure was reduced to 10(5) or less for CY treated mice. In normal mice protection was afforded on immunization with 10(7) bacteria whereas 10(3) bacteria were sufficient to protect CY treated animals. All protected mice showed a positive DH. These results demonstrate that CY treatment reduces the dose of viable bacteria tolerated for immunization 10(4) times. On the other hand after CY treatment the doses of bacteria effective on immunization for ACR and DH could be reduced in the same order of magnitude. Reduction of the CY dose resulted in a peak DH with 4 mg CY, but the protection was less than that obtained after treatment with 6 mg CY. A dissociation between ACR and DH was observed by varying the interval between immunization and challenge. In normal mice DH was preceded by ACR, with peaks at respectively 10 and 5 days after immunization. CY treatment caused a delay in the onset of the ACR, followed by an enhanced and slightly prolonged response. The effect of CY on DH consisted of enhancement and prolongation.